The objective of this proposal is to determine sequence-structure-function relationships of a protein complex called polyketide synthase (PKS), an enzyme complex comprised of 5 - 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of extender units, chain length, cyclization and reduction patterns. The focus of this proposal is to determine the crystal structures and sequence-structure-function relationship of two aromatic PKS domains, the ketoreductase (KR) and bifunctional aromatase/cyclase (ARO/CYC). KR and ARO/CYC catalyze the polyketide chain reduction and cyclization, respectively, in a highly specific manner. The central hypothesis 'or the proposed research is that we can use structure-directed mutagenesis to change the substrate specificity and enzyme activity in a predictable manner. We have formulated this hypothesis based on our preliminary results of 14 crystal structures. 30 mutants, five in vitro substrates and kinetic studies of the actinorhodin KR (actKR) and tetracenomycin ARO/CYC (tcmARO). We will pursue the following specific aims: AIM 1. Determine the Sequence-Structure-Function Relationship of Ketoreductase (KR) That Leads to its Unique Reduction Specificity, in which we will (1) determine the cocrystal structures of actKR and substrates/inhibitors in order to identify structural features important for catalysis and protein-ligand nteractions, (2) probe the active site geometry of actKR with polycarbonyl compounds as the "molecular ruler" in order to correlate different ligand binding motifs with active site geometry, and (3) identify residues important for enzyme activity, stereo-specificity and regio-specificity by kinetic assays and structure-directed mutagenesis. AIM 2. Determine the Sequence-Structure-Function Relationship of Aromatase/Cyclase (ARO/CYC) That Leads to its Unique Cyclization Specificity, in which we will (1) determine the co-crystal structures of tcmARO and inhibitors in order to dissect the protein-ligand interactions, (2) determine the crystal structures of different ARO/CYCs in order to identify important sequence-structure features for different biological functions, and (3) identify tcmARO residues important for catalysis and cyclization specificity by kinetic assays and structure-directed mutagenesis;AIM 3. Determine the Importance of Protein-Protein Interactions on the Sequence-Structure-Function Relationship between KR and ARO/CYC, in which we will correlate protein-protein interactions between PKS domains with enzyme activity and regio-specificity. The proposed research is significant, because the outcome will answer important questions about how polyketide reduction and cyclization are precisely controlled. It is also innovative by providing new information about KR and ARO/CYC at a molecular level not achieved previously. The long- term biomedical relevance is that the polyketide research community can apply the sequence-structure- function relationships determined from this proposal to diversify the population of "unnatural" natural products via protein engineering, such that a library of novel polyketides with different ketoreduction and cyclization patterns can be produced. This is expected to positively affect public health, because it will allow the development of new "unnatural" natural products that can be screened for new pharmaceutical activities. Meanwhile, the fundamental new knowledge obtained in this proposal on correlating PKS sequence-structure with the catalysis, substrate specificity and protein-protein interactions during polyketide ketoreduction and cyclization is expected to have a high impact on the natural product research communities.